Stopped-flow fluorescence of Cj1427-NADH after the addition of α-ketoglutarate. (a) Stopped-flow fluorescence traces (0.05 s) showing Cj1427•NADH conversion to Cj1427•NAD+ with various amount of α-ketoglutarate. The data were fit to a single exponential (equation 2) shown as solid lines. Data were collected using 15 μM Cj1427 (final concentration) with various concentrations of α-ketoglutarate in 50 mM HEPES/KCl buffer, pH 7.4. The concentrations of α-ketoglutarate are 0, 2.5, 5, 7.5, 10, 15, 20, 30, 50, 75, 100, 150, 200, 300, 500 μM. (b) Observed rate constant (λ1) from the 0.05 s stopped-flow data for Cj1427 reacting with α-ketoglutarate plotted versus the concentration of α-ketoglutarate. The solid line is the fit of the data to equation 3.